Natural mutations in humans and introduced mutations in mice highlight the importance of the type II cytokine, interferon-gamma (IFN-3), for control of tuberculosis (TB). How IFN-3 exerts its effects depends largely on the broad transcriptional programs it activates within Mycobacterium tuberculosis (Mtb)-infected host cells, especially macrophages. Here as many as 1,300 genes may be engaged. Our previous studies identified one member of a 47kDa Immunity Related GTPase (IRG) family - Irgm1 - as important for protection against Mtb in macrophages and in mice. Recent profiling has revealed expression of a second family of IFN-3- inducible GTPases - the 65kDa Guanylate binding proteins (GBPs) - within TB-related signatures of both Mtb- infected human and mouse tissues. Moreover, this expression appears functionally important. Preliminary studies using newly created Gbp-deficient mice find a protective role for these 65kDa GTPases during mycobacterial infection in vivo. We propose to build on these observations by pursuing two broad inter-related aims. Aim1. To understand how the IFN-3-inducible Gbps mechanistically control Mtb-infection at the level of the infected macrophage and in mice. Here we will focus on three members - Gbp1, Gbp5 and Gbp7 - that exhibit robust loss-of-function phenotypes against mycobacteria and for which initial partner interaction screens have provided some insight into the mechanisms employed. Aim 2. To determine whether these proteins also confer anti-tubercular activity within its natural human host. Here we will functionally examine the corresponding human orthologs - GBP1, GBP4 and GBP5 - in Mtb-infected human macrophages and confirm expression in human TB samples from active and latently infected individuals. Causal relationships will then be established in vivo using a new humanized mouse model amenable to GBP-specific siRNA silencing in immune cells such as macrophages. In addition, we will screen patient and kindred cohorts for loss-of-function GBP mutations that associate with mycobacterial susceptibility in collaboration with J.L Casanova. Both of these strategies should provide evidence for the IFN-3-inducible GBPs conferring protective immunity in nature. Understanding of the mechanisms employed by these new IFN-3-inducible GTPases has important implications for TB vaccine design, small drug therapy and, more recently, as novel biomarkers for disease reactivation from latency. The latter application in particular represents an urgent unmet need for the TB and infectious disease community.